US 2002O151646A1

(19) United States

(12) Patent Application Publication (10) Pub. No.: US 2002/0151646A1
Kikukawa et al. (43) Pub. Date: Oct. 17, 2002
(54) THROMBOPLASTIN REAGENT AND (30) Foreign Application Priority Data
METHOD FOR MANUFACTURING THE
SAME Feb. 5, 2001 (JP).................................. HEI 13-028828
Publication Classification
(75) Inventors: Norihiro Kikukawa, Kobe (JP);
Masahiro Okuda, Kobe (JP) (51) Int. Cl." ....................................................... C08H 1100
(52) U.S. Cl. ............................................................ 525/54.1
Correspondence Address: (57) ABSTRACT
SUGHRUE MION, PLLC
2100 PENNSYLVANIAAVENUE, N.W. This invention provides a novel thromboplastin reagent
WASHINGTON, DC 20037 (US) having high measurement Sensitivity. Thus, an effective
amount of amino acid or derivative thereof having Such a
(73) Assignee: INTERNATIONAL REAGENTS function that an ISI (international sensitivity index) of a
CORPORATION composition of thromboplastin showing an ISI of more than
1.0 is made nearer 1.0 is added whereby a thromboplastin
(21) Appl. No.: 10/062,425 having a high measurement Sensitivity is provided. To be
more Specific, Sodium glutamate is added in an effective
(22) Filed: Feb. 5, 2002 amount.
US 2002/0151646 A1
THROMBOPLASTN REAGENT AND METHOD
FOR MANUFACTURING THE SAME
BACKGROUND OF THE INVENTION
0001) 1. Field of the Invention
0002 The present invention relates to a thromboplastin
reagent used for measurement of a coagulation factor where
a novel reagent having high Sensitivity is provided and also
relates to a method for manufacturing the same.
0003 2. Description of the Related Art
0004. As a test for screening the defect of coagulation
factors in the blood of patients, Quick’s Prothrombin time
(PT) has been used. PT is also used as a monitor for the
therapy by oral anticoagulant therapy. Although measure
ment of the PT varies depending upon the property of the
thromboplastin reagent, PT of normal blood donor is 10-14
Seconds and, with regard to the PT of plasma deficient in
coagulation factors, it is preferred to use a thromboplastin
reagent which is prepared in Such a manner that the PT can
be prolonged depending upon the degree of defect of the
coagulation factor.
0005 Active thromboplastin induces the coagulation in
plasma and is composed of a lipid component and a protein
component. Protein, i.e. the tissue factor, is bonded to
membrane and is found in many various tissues. The bond
between the protein and the lipid is independent on Ca' due
to a hydrophobic interaction. Protein residue comprises
glycoprotein having a molecular weight of 43-53 kDa. One
molecule of the tissue factor is able to bond to one molecule
of a coagulation factor VII or a coagulation factor VIIa.
Bond of the coagulation factor VII/coagulation factor VIIa
to the tissue factor is dependent on Ca". A complex
comprising lipid, tissue factor and coagulation factor VIIa
cleaves a coagulation factor X to form a coagulation factor
Xa whereby blood coagulation by the activated prothrombin
is finally induced.
0006 Initiation of coagulation in plasma after 10-14
Seconds from addition of a thromboplastin reagent indicates
that the coagulation System is unhurt. An increase in the
coagulation time causes a certain disorder. The disorder
occurs as a result of too low concentration of one or more
coagulation factor(s)
0007 Thromboplastin can be extracted from many kinds
of tissueS of various animal materials. Due to the limited
availability, the cost thereof, etc., materials which are gen
erally utilized are limited and thromboplastin derived from
rabbit brain which is the most common material has a
relatively low sensitivity as compared with thromboplastin
derived typically from human tissues. Materials, extracting
method and reagent composition for thromboplastin are
important factors for determining the Sensitivity of the
reagent. Under Such circumstances, in order to improve the
Sensitivity of thromboplastin, investigation for extraction
using a nonionic detergent or the like has been attempted and
reported (Japanese Patent Laid-Open No. 03/503534).
0008 AS to another method for improving the sensitivity,
there has been reported a method where a Small amount of
protein is specifically removed. It is often that thromboplas
tins of various origins are different in terms of their Sensi
tivity indicating the defect of Specific coagulation factors. In
Oct. 17, 2002
Some cases, that is due to the fact that a Small amount of the
factor to be measured is brought into the reagent.
0009 For example, there is a report for a method of
improving the Sensitivity by a Selective inhibition of coagul
lation factor VII/coagulation factor VIIa remaining in the
thromboplastin reagent (Japanese Patent Laid-Open No.
10/330.400).
0010 For the measurement of a more correct coagulation
time, standardization of the PT measurement has been
carried out. For Such a purpose, a Standard Sample of a
thromboplastin reagent is prepared, Sensitivity of each
reagent is expressed in terms of international Sensitivity
index (hereinafter, referred to as “ISI”) on the basis of the
above and that is described for each reagent.
0011 When PT is measured using a thromboplastin
reagent, ISI value becomes low when the difference between
the PT of normal human plasma and the measured PT of
plasma deficient in coagulation factor is big while, when the
Said different is Small, ISI value becomes high. Accordingly,
the ISI value of a thromboplastin reagent having high
measurement Sensitivity is low and there has been a demand
for such a reagent. The ISI value of the international
Standard preparation of thromboplastin is 1.0.
0012 However, as mentioned already, property of a
thromboplastin reagent varies depending upon the material
composition therefor. For example, the property differs
when the material is derived from human being, rabbit or
bovine. There are many cases where there is prepared a
composition containing a thromboplastin showing an ISI
value of more than 1.0.
0013 Further, in some cases, a step of freeze-drying
treatment during the manufacturing Steps of a thromboplas
tin reagent affects the measurement of the coagulation time.
Thus, when the PT is measured using the said freeze-dried
thromboplastin reagent, there are Some cases where it is
longer than 14 Seconds even when normal plasma is used
and Such a phenomenon results from a freeze-drying pro
ceSS. The use of a thromboplastin reagent showing a value
of longer than 14 Seconds in the case of normal plasma is not
preferred in view of efficiency of the measurement and there
has been also a demand for development of a manufacturing
method whereby the damage by freeze-drying failure is
excluded.
SUMMARY OF THE INVENTION
0014. The matter to be solved by the present invention is
to provide a novel thromboplastin reagent having high
measurement Sensitivity.
0015 The present inventors have carried out an intensive
investigation by paying their attention to the ISI value and
have found that, when amino acid or amino acid derivative
is added to a thromboplastin-containing composition having
an ISI value of more than 1.0, there are some cases where the
ISI value becomes nearer 1.0. The present invention has
been achieved by addition of amino acid or amino acid
derivative having Such a function in an effective amount to
thromboplastin. It has been further investigated for the Stage
when Such an amino acid or amino acid derivative is to be
added and, as a result, it has been found that a stable
thromboplastin reagent having no reduction in the activity
by freeze-drying can be provided whereupon the present
invention has been achieved.
US 2002/0151646 A1
0016. Thus, the present invention comprises the follow
ings.
0.017. 1. A method for the manufacture of a thromboplas
tin reagent which is characterized in that, in the manufac
turing Steps of thromboplastin reagent, there is included a
Step where an effective amount of amino acid or derivative
thereof having Such a function that an ISI (international
Sensitivity index) of a thromboplastin-containing composi
tion showing an ISI of more than 1.0 is made nearer 1.0 is
added.
0.018 2. The method according to the above 1, wherein,
as the effective amount of amino acid or derivative thereof
mentioned in the above 1, it is added So as to make the final
concentration 0.01-20 w/v. '76.
0019. 3. The method according to the above 1 or 2,
wherein the amino acid or derivative thereof mentioned in
the above 1 is glutamic acid, Sodium glutamate or glycine.
0020 4. The method according to any of the above 1 to
3, wherein the step of addition of the amino acid or deriva
tive thereof is after the step for the extraction of thrombo
plastin from the material composition and is before the
freeze-drying.
0021 5. The method according to any of the above 1 to
3, wherein the step of addition of the amino acid or deriva
tive thereof is after the step for the extraction of thrombo
plastin from the material composition and for the freeze
drying.
0022 6. A thromboplastin reagent which is manufactured
by any of the methods mentioned in the above 1 to 5.
0023 7. A thromboplastin reagent, characterized in that,
there is contained an effective amount of an amino acid or
derivative thereof which has a function that an ISI (inter
national Sensitivity index) of a thromboplastin-containing
composition showing an ISI of more than 1.0 is made nearer
1.O.
0024 8. The thromboplastin reagent according to the
above 7, wherein, as the effective amount of amino acid or
derivative thereof mentioned in the above 7, it is added so
as to make the final concentration 0.01-20 w/v. '76.
0.025 9. The thromboplastin reagent according to the
above 7 or 8, wherein the amino acid or derivative thereof
mentioned in the above 7 is glutamic acid, Sodium glutamate
or glycine.
0.026 10. A kit for the measurement of coagulation time
containing the thromboplastin reagent described in any of
the above 6 to 9.
DETAILED DESCRIPTION OF THE
PREFERRED EMBODIMENTS
0027 INR (international normalized ratio) is used as a
new way of describing a PT. The INR value is calculated by
rising to ISI power of a prothrombin ratio (PR) and its
normal value is 1.0. Here, a prothrombin ratio is the ratio of
the PT of normal plasma to the PT of patient plasma and is
expressed by PR.
Oct. 17, 2002
0028 rsThe relation between INR and ISI is given by
the following formula.
INR-PR-(PT of patient plasma)/(PT of normal
plasma)
0029 rsThus, when the ISI value is high in a throm
boplastin reagent, it is a reagent where the difference
between the PT of normal plasma and the PT of plasma
deficient in a coagulation factor is little while, when the
ISI value therein is low, it is a reagent where the differ
ence between the PT of normal plasma and the PT of
plasma deficient in a coagulation factor is large. There
fore, it is believed that, when the PT changes depending
upon the amount of the coagulation factor contained
therein, amount of the coagulation factor can be mea
sured with a good precision.
0030 rSAt present, various kinds of thromboplastin
containing compositions extracted from human placenta,
rabbit brain, bovine brain, etc. are used as bulk mate
rials for thromboplastin reagent and most of such com
positions show an ISI of more than 1.0. Thus, when the
ISI is made nearer 1.0, it is possible to provide a throm
boplastin reagent having a suitable sensitivity. Such a
way of thinking is not limited to a thromboplastin
reagent derived from natural substances only but is
applicable to thromboplastin reagents prepared by
means of a recombinant technology as well. In the
present invention, a thromboplastin-containing compo
sition may be anything so far as it contains thrombo
plastin and there is no limitation for its source. For
example, thromboplastin (tissue factor)-containing com
positions not only derived from natural substances such
as human placenta, rabbit brain and bovine brain but
also prepared by means of a recombinant technology, etc.
are included.
0031 rsAn amino acid or derivative thereof having a
function of making an ISI of a thromboplastin-contain
ing composition which has an ISI of more than 1.0 nearer
1.0 is a substance which has a function of lowering the
ISI value to make nearer 1.0 when an appropriate
amount is added to a thromboplastin composition and it
stands for an amino acid or derivative thereof. Examples
of the amino acid or derivative thereof having such a
function are alanine, aminobutyric acid (hereinafter,
referred to as “ABA'), glutamic acid, glutamine, sodium
glutamate, glycine, methionine, proline, serine and
tyrosine. Preferably, they are alanine, aminobutyric acid,
sodium glutamate, glutamic acid or glycine. And a more
preferred example is sodium glutamate, glutamic acid or
glycine.
0032 rsWith regard to an effective amount of the
amino acid or derivative thereof having the above-men
tioned function, there is no particular limitation so far as
it is an amount achieving a function whereby the ISI
value is lowered to make nearer 1.0. For example, in
terms of the final concentration in a thromboplastin
reagent, a range of 0.01-20 w/v %, preferably 0.1-10 w/v.
% or, more preferably, 0.5-5 w/v 9% may be exemplified.
US 2002/0151646 A1 Oct. 17, 2002

0033 rsThe amino acid or derivative thereof having
the above-mentioned function may be added at any stage
during the manufacturing steps and there is no particu
lar limitation therefor. It is also possible that, after being
prepared as a reagent composition, the above-mentioned
effective amount is added. Especially when the amino
acid or derivative thereof having the above-mentioned
function is added with an object of preventing the reduc
tion of activity caused by the so-called freeze-drying, it is
preferred to add prior to the freeze-drying during the
manufacturing steps.
0034) rsIn addition to the freeze-dried product, the
thromboplastin reagent of the present invention may be
in a form of a liquid product or a frozen product.
activity by freeze-drying can be prevented whereby it is
possible to provide a reagent having a high Stability.
0042. In the case of a calibration plasma (AK-D), PT was
prolonged except the case where 1% alanine was added. It
Suggests that PT is elongated depending upon a reduction in
the content of the coagulation factor contained in the cali
bration plasmas (AK-A-D) and accordingly that measure
ment with better sensitivity is possible.
0043. The ISI value of each thromboplastin reagent was
smaller in all cases than the ISI value of a thromboplastin to
which no additive was added.
TABLE 1.

EXAMPLES Prothrombin Time (PT) and ISI Values

when Various Additives were Added
0035) rsThe present invention will now be further (PT: seconds)
illustrated by way of the following Examples although
Nothing 1% Glu . 2%. Glu . 3%. Glu .
the present invention is not limited thereto. INR Added Na Na Na

Example 1. AK-A 1.04 14.3 13.6 13.5 13.9

AK-B 1.92 20.4 20.8 21.7 23.4
0036) rsPT values when various concentrations of AK-C 3.07 27.1 28.4 30.1 33.5
various kinds of amino acid or derivatives thereof were
AK-D 4.37 33.3 35.8 37.6 41.5
added to thromboplastin bulk solution (derived from ISI = 1.69 148 1.4 1.3
rabbit brain) prepared by a method described in Japa
nese Patent Laid-OpenNo. 05/60,762 were measured and Nothing
then ISI value for each of the sample was determined on INR Added 1%. Ala 2%. Ala 3%. Ala
the basis of calibration plasma where an INR was pre
viously regulated. AK-A 1.04 14.3 13.5 13.9 13.6
AK-B 1.92 20.4 19.9 20.9 20.6
0037 rsMeasurement of the PT was carried out AK-C 3.07 27.1 27.1 28.2 28.3
according to a known measuring method. AK-D 4.37 33.3 33.1 34.8 35.3
ISI = 1.69 1.59 1.56 1.5
0.038 rsTo be more specific, measurement was con
ducted by the following method. Thus, each of various
amino acids or amino acid derivatives was added to a Nothing
thromboplastin bulk solution so as to make its final INR Added 1% ABA 2% ABA 3% ABA
concentration 1, 2 or 3 w/v. '76, then a calcium salt was AK-A 1.04 14.3 13.5 13.6 13.5
added thereto to make its final concentration 10 mM and
the mixture was freeze-dried to give a thromboplastin AK-B 1.92 20.4 20.3 2O.9 21.0
sample. Calibration plasma (0.05 ml) was pipeted and AK-C 3.07 27.1 27.4 27.8 28.4
incubated at 37° C. for about 1 minute. To this was added AK-D 4.37 33.3 34.2 34.6 35.3
0.1 ml of a thromboplastin reagent solution which was ISI = 1.69 1.54 1.54 1.49
previously reconstituted in pure water and mixed with
calcium in a concentration of 10 mM incubated at 37° C.
and then PT was measured using a Coagrex-700 (an 0044. Each of AK-A, B, C and D is calibration plasma
automated coagulation analyzer; manufactured by Shi and has an intrinsic INR value.
madzu).
0039) rSAn ISI value of each thromboplastin sample 0045 ISI value for each sample was calculated from the
solution was determined from the measured PT value measured PT value and the calibration plasma INR value for
and the INR value of the calibration plasma and influ each Sample.
ence of each additive on ISI was tested.
Example 2.
0040. The result was shown in Table 1.
0041 As a result, with regard to a calibration plasma 0046 PT was measured for the case where sodium
(AK-A), PT was shortened in all cases where a thrombo glutamate was added as an additive to a thromboplastin bulk
plastin reagent to which amino acid or derivative thereof Solution to Such an extent that its final concentration was 1,
was added was used as compared with the case of the use of 2, 3, 4 or 5 w/v. '76 and then an ISI value was determined.
thromboplastin reagent to which no additive was added.
Thus, it is likely that, when various additives are added to a 0047. Measurement of PT and calculation of ISI value
thromboplastin bulk reagent, the So-called reduction in the were carried out in the same manner as in Example 1.
US 2002/0151646 A1 Oct. 17, 2002

0048. The result was shown in Table 2.

TABLE 2
Prothrombin Time (PT) and ISI Values when Various
Concentrations of Sodium Glutamate were Added
PT: seconds

Nothing
INR Added 1% Glu Na 2%. Glu . Na 3% Glu . Na 4% Glu Na 5% Glu . Na
AK-A 1.04 14.7 14.3 14.O 14.O 14.8 15.5
AK-B 1.92 20.5 21.6 22.4 24.7 25.9 28.7
AK-C 3.07 28.0 30.2 31.5 35.5 38.8 4.1.8
AK-D 4.37 35.2 38.6 41.2 46.4 SO4 57.5
ISI = 1.64 1.44 1.33 1.2 1.16 1.1

0049. Each of AK-A, B, C and D is calibration plasma
and has an intrinsic INR value.
0050 ISI value for each sample was calculated from the
measured PT value and the calibration plasma INR value for
each Sample.
0051. The ISI value for each thromboplastin sample
became Small depending upon the concentration of Sodium
glutamate and an improvement in the Sensitivity of each
thromboplastin Sample was noted.
0.052 On the other hand, the PT for the amended plasma
(AK-A) was shortest when 2 w/v % or 3 w/v % of sodium
glutamate was added to give a thromboplastin reagent
having a high stability.
0.053 As fully illustrated hereinabove, it is now possible
to provide a thromboplastin reagent having a high measure
ment Sensitivity and a high Stability when, in the manufac
turing Steps of thromboplastin reagent, there is included a
Step where an effective amount of amino acid or derivative
thereof having Such a function that an ISI (international
Sensitivity index) of a composition of thromboplastin show
ing an ISI of more than 1.0 is made nearer 1.0 is added.
What is claimed is:
1. A method for the manufacture of a thromboplastin
reagent which is characterized in that, in the manufacturing
Steps of thromboplastin reagent, there is included a step
where an effective amount of amino acid or derivative
thereof having Such a function that an ISI (international
Sensitivity index) of a thromboplastin-containing composi
tion showing an ISI of more than 1.0 is made nearer 1.0 is
added.
2. The method according to claim 1, wherein, as the
effective amount of amino acid or derivative thereof men
tioned in the above 1, it is added So as to make the final
concentration 0.01-20 w/v. '76.
3. The method according to claim 1 or 2, wherein the
amino acid or derivative thereof mentioned in claim 1 is
glutamic acid, Sodium glutamate or glycine.
4. The method according to any of claims 1 to 3, wherein
the step of addition of the amino acid or derivative thereof
is after the step for the extraction of thromboplastin from the
material composition and is before the freeze-drying.
5. The method according to any of claims 1 to 3, wherein
the step of addition of the amino acid or derivative thereof
is after the step for the extraction of thromboplastin from the
material composition and for the freeze-drying.
6. A thromboplastin reagent which is manufactured by
any of the methods mentioned in any of claims 1 to 5.
7. A thromboplastin reagent, characterized in that, there is
contained an effective amount of an amino acid or derivative
thereof which has a function that an ISI (international
Sensitivity index) of a thromboplastin-containing composi
tion showing an ISI of more than 1.0 is made nearer 1.0.
8. The thromboplastin reagent according to claim 7,
wherein, as the effective amount of amino acid or derivative
thereof mentioned in claim 7, it is added So as to make the
final concentration 0.01-20 w/v. '76.
9. The thromboplastin reagent according to claim 7 or 8,
wherein the amino acid or derivative thereof mentioned in
claim 7 is glutamic acid, Sodium glutamate or glycine.
10. A kit for the measurement of coagulation time con
taining the thromboplastin reagent described in any of
claims 6 to 9.